Project Summary Targeted therapies have shown great promise in the treatment of cancer. High-throughput screening (HTS) campaigns have often relied on assays using labeled ligands or enzyme substrates. Artifacts associated with labeling have led to the erroneous identification of compounds that act in on the labeled substrate rather than the intended target. A label-free, solution-based HTS method will facilitate identification of compounds acting specifically on the intended targets. The goals of this proposed project are to advance development and validate this calorimetry platform as an accessible approach for high throughput ITC analysis, demonstrating its advantages to study a variety of cancer therapeutic targets. To achieve this goal, we will improve mixing of reagents in droplets, multiplex temperature readout from individual droplets, automate fluidic handling and data analysis, and validate the applicability of the improved technology to study: 1) SH2 domain/phosphopeptide interactions, 2) spleen tyrosine kinase (Syk) enzyme activity, and 3) Bromodomain/acetylated histone peptide interactions, further applied in small molecule screening. The realization of the spectro-optical calorimetry technology platform will enable full thermodynamic characterization of binding and enzymatic reactions at the throughput of 1536-well plate assays but with significantly reduced assay development time and larger numbers of repeats and analyte concentrations. The combination of increased throughput and reduced sample consumption will change the way researchers view calorimetry: rather than being seen as a technique for a limited number of high-value measurements, it will be viewed as a primary screening method for cancer therapeutic targets.